Forcefully-Aerated Bioreactors Without Mixing

19 Pages·2017·0.32 MB·English

7 Group II Bioreactors: Forcefully-Aerated
Bioreactors Without Mixing
David A. Mitchell, Penjit Srinophakun, Nadia Krieger, and Oscar F. von Meien
7.1 Introduction
This chapter addresses the design and operation of SSF bioreactors under condi-
tions where forced aeration is used but the substrate bed is not mixed. Typically
these bioreactors are referred to as packed-bed bioreactors. This mode of operation
is appropriate for those SSF processes in which it is not desirable to mix the sub-
strate bed at all during the fermentation due to deleterious effects on either micro-
bial growth or the physical structure of the final product.
The characteristics of this mode of operation also apply to the static phases of
forcefully-aerated bioreactors that are mixed once every few hours. The operation
of such bioreactors will be discussed in Chap. 10; suffice to say for the moment
that during the static phase they will act as packed-bed bioreactors, and therefore
the principles developed in the present chapter will apply to this static phase.
7.2 Basic Features, Design, and Operating Variables for
Packed-Bed Bioreactors
The basic design features of a packed-bed bioreactor have been already presented
in Sect. 3.3.1. Figures 7.1 and 7.2 show these features in more detail. Some possi-
ble variations in the design include:
(cid:120)(cid:3) the column may have a cross section other than circular.
(cid:120)(cid:3) the column may lie horizontally, or for that matter, at any angle. This alters the
relative directions of the forces due to gravity and air pressure.
(cid:120)(cid:3) the column may be aerated from either end. For a vertical column, the air may
enter the bed from either the top or the bottom. Aerating from the top avoids
the fluidization of particles at high air velocities, but will contribute to bed
compaction since the air flow is in the same direction as gravity.
78 7 Group II Bioreactors: Forcefully-Aerated Bioreactors Without Mixing
(cid:120)(cid:3) the column may have a perforated tube inserted along its central axis, allowing
an extra air supply in addition to the end-to-end aeration (Fig. 7.1(b)). How-
ever, this will only be effective for very small bioreactor diameters.
(cid:120)(cid:3) the column may be water-jacketed or heat transfer plates may be inserted into
the bed. In this chapter, packed-bed bioreactors with internal heat transfer
plates will be referred to as (cid:147)Zymotis packed-beds(cid:148), using the name coined by
Roussos et al. (1993), while those lacking such plates will be referred to as
(cid:147)traditional packed-beds(cid:148) (Fig. 7.2).

(a) (b)
(c) (d)

Fig. 7.1. Basic design features of packed-bed bioreactors and possible design variations.
(a) A simple (cid:147)traditional(cid:148) packed-bed design. (b) A packed bed with a perforated tube in-
serted along its central axis: The benefits of this will only be apparent if the bed is relatively
thin or, in a wide bed, if many perforated tubes are inserted. This is due to the fact that the
forced aeration in the axial direction will tend to force the radial flow to follow the axial di-
rection also. (c) Radial flow packed-bed: The advantage of this design is that, compared to
a column of the same dimensions, the distance of flow through the bed is decreased. It is
similar to the use of a wider (cid:147)traditional(cid:148) packed bed with a lower bed height. (d) A (cid:147)short-
wide(cid:148) packed-bed
7.2 Basic Features, Design, and Operating Variables for Packed-Bed Bioreactors 79
(a)
s
n
os
diting
ndi
on
Cu
wDiedstihg no:f Htheei gbhetd and erating: he surro
Opn t
i perforated
base plate
exploded
view
Operating: The flow rate and the chamber under
temperature of the inlet air the bed may be
(the air should be saturated) filled with packing to
promote an even
distribution of air
(b) exhaust air
cooling
water in
and out
e
c
a
p Operating: Temperature and
s
d flow rate of the cooling water
a
e
H
Design: the height of the heat
transfer plates.
Operating: the height of the
s substrate bed
e
at d
pl be Design: The overall width of
ansfer strate tbheet wbeioerne ahcetaotr tarannds tfheer pslpaatecisng
r b
at t su Operating: The flow rate and
e temperature of the inlet air
H
inlet (the air should be saturated)
air

Fig. 7.2. Basic design and operating features of (a) traditional packed beds and (b) the Zy-
motis packed-bed with internal heat transfer plates of Roussos et al. (1993)
80 7 Group II Bioreactors: Forcefully-Aerated Bioreactors Without Mixing
Taking the most common design, namely a vertical column in which the bed is
aerated from the bottom and without any internal perforated tubes, the available
design variables for a packed-bed are (Fig. 7.2):
(cid:120)(cid:3) the presence or absence of a cooling jacket or internal heat transfer plates;
(cid:120)(cid:3) the height and width of the bioreactor. The height to diameter ratio can vary
over quite a wide range;
(cid:120)(cid:3) if internal cooling plates are used, their height and the spacing between them.
The available operating variables are (Fig. 7.2):
(cid:120)(cid:3) the aeration rate;
(cid:120)(cid:3) the temperature of the inlet air;
(cid:120)(cid:3) the temperature of the (cid:147)surroundings(cid:148) (which might be cooling water).
In a static bed, the relative humidity of the inlet air is not a useful operating
variable. The problem is that it is not practical to add water into an unmixed bed in
such a way as to distribute it evenly amongst the substrate particles; therefore
evaporative water loss must be minimized. If the air entering the bed were not
saturated with water, this unsaturated air would promote evaporation and dry out
the bed, eventually decreasing the water activity to values unfavorable for growth
and product formation. In order to minimize evaporation, saturated air must be
supplied at the air inlet, which removes manipulation of the inlet air humidity as
an available operating variable. Note that the use of saturated air does not prevent
evaporation from occurring within the bed (see Fig. 4.3), but it does minimize
evaporation compared to the use of unsaturated air. As will be discussed in Chap.
10, it is possible to replenish water during the mixing events of intermittently
mixed beds, in which case unsaturated air can be used to aerate the bed.
At large scale, water-jacketing of the side walls of the bioreactor is not a good
idea for the traditional design, since the water jacket will influence only the outer
20 cm or so of the bed. If cooling surfaces are to be used, then the internal cooling
plates used in the Zymotis bioreactor will be more effective, as long as they are
reasonably closely spaced. Optimum spacing of the plates will be discussed later.
Another option for cooling surfaces is given by the (cid:147)Prophyta(cid:148) and (cid:147)PlaFractor(cid:148)
designs (Fig. 7.3), two bioreactors that use a number of thin beds coupled with
cooling plates oriented normal to the air flow (L(cid:252)th and Eiben 1999; Suryanarayan
and Mazumdar 2000; Suryanarayan 2003). The difference between the two biore-
actors is that in the Prophyta design the same air passes through each successive
bed while in the PlaFractor design the air is introduced separately into each bed.
Important phenomena that are affected by the values chosen for the design and
operating variables are:
(cid:120)(cid:3) the axial and radial temperature gradients in the bed. In packed-beds it is im-
possible to prevent temperature gradients from arising within the bed, so the
aim is generally to minimize the size of any temperature gradients.
(cid:120)(cid:3) the evaporation of water from the bed. Efforts must be made to minimize
evaporation in order to prevent the bed or parts of the bed from drying out.
7.3 Experimental Insights into Packed-Bed Operation 81
(cid:120)(cid:3) the pressure drop through the bed. This will depend on the bed height and the
degree to which the organism fills the inter-particle spaces, with the resulting
pressure drop affecting the design of the aeration system and its operating costs,
and maybe placing a limit on the bed height that can be used.
In general, O supply to the particle surface will not be considered in the selec-
2
tion of design and operating variables. The aeration rates that are chosen on the
basis of heat removal considerations will typically be high enough that sufficient
O supply is ensured. However, note that problems such as channeling are possi-
2
ble, in which O transport to large parts of the bed can be limiting (Fig. 7.4).
2
Channeling is discussed in more detail in Sect. 7.3.3.5.
This chapter explains what is known, on the basis of experimental studies,
about how these design and operating variables influence bioreactor operation.
Later, Chap. 24 will show how mathematical models can be used to explore fur-
ther the design and operation of packed-beds.
7.3 Experimental Insights into Packed-Bed Operation
This section presents and discusses the knowledge that experimental work has
given firstly into the phenomena that occur within packed-bed bioreactors, and
secondly into the operability of this type of bioreactor.

(a) (b)

Fig. 7.3. The use of heat transfer surfaces normal to the air flow direction within packed
beds (a) as used in the Prophyta bioreactor (L(cid:252)th and Eiben 1999) and (b) as used in the
PlaFractor bioreactor (Suryanarayan and Mazumdar 2000; Suryanarayan 2003). In each
case the substrate beds are in light gray and the heat transfer plates are in dark gray. The
white regions represent empty spaces for air flow. Solid arrows represent the flow of cool-
ing water and dashed arrows represent the flow of air
82 7 Group II Bioreactors: Forcefully-Aerated Bioreactors Without Mixing
(a) (b) (c)
(d) tensile force of inter-
particle hyphal bridges
heat removal from much
of the bed can be limited
to conduction
O supply to much of
2
the bed can be limited
to diffusion
more resistance to less resistance to
flow encountered flow encountered

Fig. 7.4. The phenomenon of channeling. (a) The desirable situation, with uniform flow
across the whole cross section of the bed. (b) Preferential flow between the bed and the
wall in the case in which the bed pulls away from the wall. (c) Preferential flow through a
crack in the bed. (d) Microscale view of a channel, showing how the preferential flow
through the channel arises due to two sources of resistance to flow through the bed of parti-
cles, namely the tortuous path through the bed and the fact that the space between the parti-
cles is partially filled with biomass. Note that in extreme cases of channeling, there may be
no bulk flow through the inter-particle spaces, with mass transfer being limited to diffusion
and heat transfer to conduction
7.3.1 Large-Scale Packed-Beds
SSF bioreactors are only rarely operated at large scale as packed-beds throughout
the entire cultivation period, although related intermittently stirred designs have
been used quite successfully (see Chap. 10). Static packed-bed operation has been
used at large scale in the production of koji, although details of the operation and
performance of the bioreactors involved are not available. Only very brief and
general descriptions are available. A simple design (Fig. 7.5) has a capacity for
1000 kg of koji, and has no special devices for substrate handling. Also, it is not
designed for fully aseptic operation (Sato and Sudo 1999).
7.3 Experimental Insights into Packed-Bed Operation 83
these regions
sealed with cloth
h
eg
orhi m
mm e
bed not han 20 c ation syst
t r
a
p
e
pr
r
ai

Fig. 7.5. Simple packed-bed of the type used in the koji industry for soy sauce production
by Churitsu Industry Co. Ltd., Tokyo, Japan. Bioreactors of this type have capacities of up
to one ton of substrate. This is a simplified version of a diagram presented by Sato and
Sudo (1999)
7.3.2 Pilot-Scale Packed-Beds
Roussos et al. (1993) developed a pilot-scale packed-bed bioreactor with internal
heat transfer plates, called the (cid:147)Zymotis(cid:148) bioreactor (Fig. 7.2(b)). The outer casing
was acrylic, and it was 65 cm high, 50 cm wide, and 40 cm deep from front to
back. This gave a total volume of 130 L, with a working capacity of 100 L. The
aeration rate was varied from 0.1 to 0.2 L h-1 g-dry-substrate-1.
The stainless steel heat transfer plates were 60 cm high, 38 cm wide (fitting
within the 40 cm front to back depth of the outer casing), and 0.46 cm thick. There
were 10 of these, and they occupied a volume of 9.44 L of the bioreactor vessel.
Each heat exchanger plate contained serially placed tubes through which water
was circulated. The bioreactor was designed to be flexible in that the number of
heat transfer plates inserted and the spacing between them could be changed as
desired. This bioreactor was emptied by raising the whole bioreactor and letting
the substrate bed fall out of the bottom, although at large scale this might not be
feasible.
Substrate loadings from 4 to 12 kg dry substrate matter (15 to 55 kg substrate
on a wet basis) were tested, but detailed performance data was not provided, only
the final enzyme levels. Heating of the circulating water was required during the
first 10 h of fermentation, after which cooling was necessary. Thermistors placed
in different locations were used to control the temperature of the cooling water.
Uniformity of growth and absence of temperature gradients was claimed when the
gap between plates was no larger than 5 cm, but experimental results showing this
were not presented (Roussos et al. 1993). The water content also remained close to
the original value, increasing by only 5%.
84 7 Group II Bioreactors: Forcefully-Aerated Bioreactors Without Mixing
7.3.3 Laboratory-Scale Packed-Beds
The use of very small and thin packed-bed bioreactors in laboratory-scale studies
of growth kinetics will be discussed in Chap. 15. A range of slightly larger biore-
actors, typically up to 30 cm high and from 5 to 15 cm diameter, have been used
to investigate how macroscale transport phenomena can influence bioreactor per-
formance. The limitation of growth by transport phenomena is possible even at
this small scale, as demonstrated in the following subsections.
7.3.3.1 Axial and Radial Temperature Gradients in Static Beds
The gas flow pattern within the bed of a packed-bed bioreactor that does not suffer
from channeling problems is probably closest to plug flow with axial dispersion
(see Fig. 4.6). However, studies have neither been done to confirm this nor to
quantify the degree of axial dispersion. This plug-flow of the gas phase has impli-
cations for the operation of packed-beds. Firstly, the inlet end tends towards the
inlet air temperature but, due to the lack of mixing and the unidirectional air flow,
the temperature of the air increases as it flows along the bed towards the outlet end
(Fig. 4.3(b)). One of the major challenges in designing and operating large-scale
packed-beds will be to avoid excessive axial temperature gradients.
The increase in the temperature of the air as it flows through the bed increases
the water-carrying capacity of the air and therefore evaporation will occur. Note
that evaporation will occur even if saturated air is used at the air inlet (Fig. 4.3(c)).
In general, conduction along the axis in the direction of the air flow will be
negligible compared to the convective and evaporative heat removal (Gutierrez-
Rojas et al. 1996). The contribution of conduction normal to the direction of the
air flow will depend on the design of the packed-bed. In traditional packed-beds
that have diameters of the order of a few centimeters and in the Zymotis design, it
can make a significant contribution, and there can be significant temperature gra-
dients normal to the air flow. In contrast, in large-scale packed-beds, which might
typically have diameters of the order of 1 m or more, the amount of energy re-
moved from the bed by transfer through the side walls is likely to be small, even if
the bed is water-jacketed. Various studies have been done that show how the ap-
pearance of axial and radial temperature gradients depends on the design and op-
eration of the bioreactor. These are described below.
Temperature gradients in thin bioreactors. Saucedo-Castaneda et al. (1990)
used a bioreactor of 6 cm diameter, containing a bed 35 cm high. Further, the col-
umn was immersed in a constant temperature waterbath at 35(cid:176)C. They noted a
steep temperature gradient in the first 5 cm along the axis of the bed, where the
temperature increased by up to 12(cid:176)C (Fig. 7.6). In contrast, in the upper 30 cm of
the bed, the maximum increase in temperature along the axis was approximately
3(cid:176)C. Note that, at some times and in some regions, the temperature actually de-
creased with axial distance, which might be related to evaporative cooling. Howe-
ver, Saucedo-Castaneda et al. (1990) did not measure water contents in the bed, so
it is not possible to confirm this. The axial temperature in the upper 30 cm of the
7.3 Experimental Insights into Packed-Bed Operation 85
column did not remain constant; rather it increased with time over the period of 15
to 26 h. In contrast to the axial temperature gradients, the radial gradients were
quite steep: at the time of peak heat production, there was an 11(cid:176)C difference be-
tween the central axis and the bioreactor wall, which represents a distance of only
3 cm. These results suggest that in the case of thin bioreactors a significant
amount of heat is removed through the side walls.

26 h 19 h 15 h
30
20
bed
height
(cm)
10
air
0
50 45 40 35
temperature ((cid:176)C) along the central axis
50
C) 45
((cid:176) 20 h
e
ur
erat 40 15 h
p
m
e
t 35
0 1 2 3
radial position (cm)

Fig. 7.6. Radial and axial temperature gradients at various times within a thin packed-bed
bioreactor when Aspergillus niger was cultivated on cassava chips (Saucedo-Castaneda et
al. 1990). The radial temperature gradient was determined at approximately mid-height in
the bed. The superficial velocity of the air was 1 cm s-1. Adapted from Saucedo-Castaneda
et al. (1990) with kind permission from John Wiley & Sons, Inc.
86 7 Group II Bioreactors: Forcefully-Aerated Bioreactors Without Mixing
Temperature gradients in wide or insulated bioreactors. The studies of Ghild-
yal et al. (1994), Gowthaman et al. (1993a, 1993b), and Weber et al. (2002) allow
insights into heat transfer in wider bioreactors. The bioreactor of Ghildyal et al.
(1994) and Gowthaman et al. (1993a, 1993b) was 15 cm in diameter with a
34.5 cm bed height, while that of Weber et al. (2002) was 20 cm in diameter with
a 50 cm bed height. Note that the bioreactor of Weber et al. (2002) was insulated
on the sides, in order to mimic the situation at large scale where heat transfer
through the walls makes a negligible contribution to heat removal. Note also that
different organisms were used in the various studies, with quite different optimal
temperatures for growth, so the actual temperatures involved are quite different.
Weber et al. (2002) measured the temperature as a function of time at various
axial positions (Fig. 7.7(a)). At all heights, there was a temperature peak, whose
maximum value occurred around day 4, with the height of the peak (that is, the
maximum temperature reached) increasing with bed height.
Ghildyal et al. (1994) presented results that show the effect of the aeration rate
on the axial temperature profile. Three different experiments were done, with air
flow rates of 5 L min-1, 15 L min-1, and 25 L min-1. The temperature was moni-
tored at the central axis at mid-height in the bed. The height of the temperature
peak increased as the air flow rate was decreased (Fig. 7.7(c)). They interpolated
their data points to obtain a three-dimensional graph of the peak temperature ob-
tained as a function of both bed height and air velocity (Fig 7.7(d)). In general
terms, the temperature appears to increase linearly with increase in bed height and
also to increase linearly with decrease in the air flow rate, except at the lowest bed
height, where the peak temperature first increased only slowly as the air flow rate
was decreased, but then shot up steeply at low air flow rates.
7.3.3.2 Oxygen Gradients in Static Beds
The convective flow of air will also lead to axial concentration gradients of O and
2
CO , although these are not likely to be important in controlling the performance
2
of the bioreactor. Outlet gas concentrations remain reasonably high, above 18%
(v/v), even at low airflow rates (Fig. 7.8) (Gowthaman et al. 1993a,b). Excessive
temperatures are always likely to be a greater problem than the supply of O to the
2
particle surface within packed-bed bioreactors.
7.3.3.3 Evaporation and Water Gradients in Packed-Beds
As pointed out in Sect. 7.2, it is impossible to prevent evaporation from occurring
in packed-bed operation, even if the air supplied to the bed is saturated. Ghildyal
et al. (1994) and Gowthaman et al. (1993a,b) showed that the rate at which bed
drying occurred depended on the position within the bioreactor and the air flow
rate (Fig. 7.9).
Looking at the effect of air flow rate on the temporal variations in water content
at each bed height, the key observations are (Fig. 7.9(a)):